Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus.

0022-538X/86/080308-10$02.00/0

Copyright C) 1986, American Society forMicrobiology

Molecular

Basis for

Interference

of Defective Interfering Particles of

Pseudorabies Virus with Replication of

Standard Virus

CAROL ANN WU, LINDA HARPERANDTAMARBEN-PORAT*

Department ofMicrobiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received 10February 1986/Accepted22April 1986

Serialpassage ofpseudorabiesvirus(PrV) at high multiplicity yields defective interfering particles (DIPs), butthe sharpcyclical increases and decreases in titer of infectious virus that areobserved upon continued passage at highmultiplicityof most DIPsof other viruses are not observed with DIPs of PrV (T. Ben-Porat and A. S. Kaplan, Virology 72:471479). Wehavestudied thedynamics of the interactions of the virions present inapopulation of

DIPs

toassessthecisfunctionsfor which thegenomes of the DIPs are enriched. The defective genomes present in onepopulationofDIPs, [PrV(1)42], replicatepreferentiallyoverthenondefective genomes present in that virion population at early stages of infection, indicating that the DIP DNA is enriched for sequences that can serve as origins of replication at early stages of infection. This replicative advantage of the DIPDNA is transientand disappears at later stages of infection. The defective DNA does not appear to be encapsidatedpreferentiallyover thenondefective DNA present in this virionpopulation, which might indicate that it is not enriched for cleavage-encapsidation sites. However, the nondefective DNA in the DIP virion

populationhas become modified and has acquiredreiterations ofsequences originating from the end of the unique long (UL) region of the genome. Furthermore, both the infectious and defective genomes present in the DIPpopulation compete for encapsidation moreeffectivelythan do thegenomes of standard PrV. These results indicate that the defective genomes in the population of virionsstiudiedareenriched not only for an origin of replication but probably also forsequences necessary for efficient cleavage-encapsidation. Furthermore, the nondefective genomes present in this population of DIPs have also been modified and have acquired theability

tocompete with the defective genomes forcleavage-encapsidation. Serialpassage athighmultiplicity ofmanyviruses,

includ-ingtheherpesviruses, resultsintheemergence ofdefective

interfering

particles (DIPs) which contain altered genome structures. These DIPs, although unable to replicate in the

absence of standard helper virus, have agrowth advantage

over it. Theidentity of theDIPs is of interest because their

emergence hasobviousimplications concerningthemodeof replication andencapsidation of the virus genome.

That DIPsofherpesvirus containgenomes thatconsist,at

least inpart, of tandemrepeats of restricted regionsofthe

viralgenome inhead-to-tail alignment has beenrecognized for some time from theirpartial denaturation patterns (12, 24). Since populations ofDIPs

becomne

enrichedfor virions

whose genomes probably canreplicate and beencapsidated,

commonfeatures oftheserepeated sequences may, in

prin-ciple,

include

origins ofreplication, as well as recognition sites for cleavage and encapsidation. Indeed, it has been

established that the DIPs of herpes simplex virus (HSV) containgenomes composed of reiterated segments ofDNA thatinclude an origin ofreplication, as well as the signals required forefficient

cleavage-encapsidation

(1,

26, 27,

29). Because

these

DIPs have a clear replicative advantage,

cyclical increases and decreases in

the

level of infectious virus areobserved as the populations of DIPs arepassaged athigh multiplicity.

Thegenomes present in twoindependentlyderived popu-lations of DIPs of pseudorabies virus (PrV)

have

been

partially characterized. The two

populations

differ in se-quence composition (22). PrV DIP DNA contains reitera-tions of a limited set ofsequences of the standard genome (2, 24) which are composed of segments of DNA originating from noncontiguous regions of the standard genome that

* Correspondingauthor.

have become covalently linked (22). One of these popula-tions, [PrV(2)], consists ofDIPs withgenomesthat contain

an intact immediate-early gene that is expressed in the

absence of helper virus. Coinfection of cells with this

popu-lationofDIPsandhelper virus causestheoverproduction of

someotherviral

proteins

aswell(23). Infection of cells with the otherpopulation of DIPs, [PrV(1)], on the otherhand, does not detectably affect viral

protein

synthesis (23).

Nev-ertheless, thebiological properties of thetwopopulations of

DIPs

appear to be quite similar (4). In both, there is a

reduction of approximately

90%

in the total number of

virionsproduced. Both populations ofPrVDIPsbehavein a

significantly different manner from most DIPs of other herpesviruses (for example, HSV and equine herpesvirus [11, 13,

28]).

Thus, the DIPs of PrV accumulate rather

slowly; only after 25 passages at high multiplicity can a

significant dropin the titerof infectiousvirus beobserved

(2,

4). Furthermore, thesharpcyclical increasesand decreases in the titersof infectiousvirusobservedupon serialpassage

ofpopulations ofDIPsofHSVandequine herpesvirus (11, 13, 28), for example, do not occur when PrV DIPs are

passaged; after an initial drop in the titer ofabout

102

by

passage30, only slightfluctuations in the titerareobserved

upon continued passage (4). The studies presented in this paper were undertaken to

clarify

thedynamics of the inter-actions between the

defective

and nondefective virions present in thepopulations of DIPs.

MATERIALS ANDMETHODS

Virus and cell culture. The preparation of PrV and culti-vation of primary rabbit kidney (RK) cells has been de-scribed previously (19). Two populations of PrV DIPs

[PrV(1)

and PrV(2)] were generated by independent serial passageathighmultiplicityof the standardPrV(Ka)strain in 308

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RKcells (4).Populations ofDIPsatvariouslevelsofpassage were used, startingwith passage 33 [PrV(1)33] andgoingto passage64 [PrV(1)64].

Media and solutions. The following media were used: Eagle synthetic medium (9) plus 5% dialyzed calf serum (EDS); EDS withoutP04(EDS-PO4); EDS-PO4plus 20 ,ugof 5-fluorouracil (FU) and 5 F.g of thymidine per ml (EDS-P04+FU); EDS-PO4 with 20 ,ug ofFU, 10 ,ug of bromode-oxyuridine (BUdR), and5 ,ugofdeoxycytidine (CdR) perml

(BUdR medium); self-digested (nuclease-free) pronase (2 mg/ml) in 0.02 M NaCl-0.02 M Tris (pH 7.3) (pronase

solution); and 0.15 MNaCl-0.015 Msodium citrate (pH 7.2) plus 4% sodium lauryl sarkosinate-97 (lx SSC-4%

sarkosyl).

Enzymes and chemicals. All restriction enzymes were obtained from Bethesda Research Laboratories, and diges-tions were performed accordingto the specifications ofthe

supplier. Inorganic 32p (carrierfree) and [a-32P]dCTP were purchased from ICN Pharmaceuticals, Inc., FUand BUdR

wereobtained from Calbiochem-Behring, and

[3H]thymidine

and [3H]deoxycytidine

(3H-CdR)

were obtained from Schwarz/Mann.

Labeling and purification of replicating virus DNA. RK

cells were starved of phosphate by incubation in EDS-P04+FU for 48 h, a procedure which also inhibits cellular DNA synthesis(18). The cells werethen incubated for 24 h in the same mediumcontaining

32p

(100

p.Ci/ml)

tolabel the

nucleotide pools. The cultures were infected and further incubated in BUdRmediumcontaining

32p

(100 ,uCi/ml). At theindicated times, the cultureswereharvested by scraping

the cells into lx SSC-4% sarkosyl. The samples were heated at 60°C for 15 min, an equal volume of pronase

solutionwasadded, and the sampleswere incubatedfor 2 h at 37°C. Viral DNA was separated from cellular DNA by isopycnic centrifugation in CsCl, with

[3H]thymidine-containing and

[3H]BUdR-substituted

viral DNA as mark-ers, as described

previously

(6). DNA fractions were col-lecteddropwise (2 dropspertube),theradiolabeledmarkers werelocalized, theappropriate fractions werecollected and

dialyzed,and the DNA was ethanol

precipitated.

Plaquepurification. Viruswas plaque assayedonRKcells grown on 90-mm petri plates. Plaques were

picked

from

plates containing not more than 20 plaques each, and the

virus wasamplified.

Purification of virionsandextraction of viralDNA. Virions were purified and viral DNA was extracted as described previously (2, 5).

RestrictionenzymedigestionandgelelectrophoresisofDNA fragments.Digestionand agarosegel

electrophoresis

ofvirus DNAwerecarriedout asdescribed

by

Rixonand Ben-Porat (22). Filterstrips,towhichrestrictionfragmentsof PrVwere fixed, were prepared by the method of Southern (25).

Nick translation of clonedPrV DNArestriction fragments.

PrV DNA restriction fragments cloned in pBR325 as

de-scribed previously (20) were nick translated by the method

ofRigby etal. (21).

RESULTS

Lack of DIP DNApreferential encapsidation. The genera-tion and amplification of DIP DNA may result from a replicative advantage of thisDNA, from a selective advan-tageof this DNAatthelevelofencapsidation, orfrom both. To determine whether preferential encapsidation of DIP DNA occurs (i.e., whether this DNA may be enriched for

cleavage-encapsidation signals), we

compared

therestriction

patternsof the viral DNAthatis

packaged

into virionswith

thoseoftheviral DNA that accumulateswithintheinfected cells.

PrV DNA is synthesized in excess, and only part of it becomes encapsidated (3, 17). Furthermore, restriction en-zyme digestion of PrV DIP DNA generates aberrantly

mi-grating DNA fragments thatare absent from the

digests

of

standard PrV DNA (22). Preferential

encapsidation

ofDIP DNA should therefore be

readily detectable,

because the presence ofthese aberrant DNA bands is

diagnostic

ofthe presence ofDIP DNA. This procedurecanbe usedto study notonlytheevolution ofthe DIP upon passageofthevirions

but also to compare the

efficiency

of

encapsidation

ofDIP DNA with that of standard DNA.

Therestrictionpatternofvirus DNA that hadaccumulated

in cells infected with

populations

of PrV(1) at different

passage levels was

compared

with that of DNA that had become

encapsidated

and was present in

preparations

of

purified

virions isolated from thesecells

(Fig.

1). No

signif-icantpreferential

encapsidation

ofDIP DNAat anylevel of

passagewas

detectable;

the viral DNA that hadaccumulated within the infected cells during the infectious cycle had a restrictionpatternsimilartothatofthe viral DNA present in the

purified

virions isolatedfromthese cells. Therestriction

patternof the viral DNA

changed, however,

uponcontinued

passageof the virions. In

particular,

enrichmentforBamHI

fragments

10aand 12b

(generated

from defective

DNA)

was observed

during early

passage levelsat

high multiplicity. By

passage 64, two defective DNA

fragments,

lla and

12b,

made up most of the DNA in the virion

population. Thus,

although the defective DNA (as

exemplified by fragment

12b)didnotappeartohavea

significant

selective

advantage

overnondefectiveDNA presentinthe DIP

population

atthe levelof

encapsidation,

itwasneverthelessenriched forupon

high-multiplicity

passage. It

appeared possible,

therefore,

thatenrichmentof the DIP DNA in the

population

occurs as aresult of

preferential

replication.

Time course of synthesis of DIP DNA. To determine

whether DIP DNA hasa

replicative advantage

either

during

the first or

during

later rounds of DNA

replication,

we

followed the time course of its

synthesis

relative to that of

the nondefective DNA present in the

population

of DIPs.

Phosphate-starved RKcellswere

pretreated

with FU and

thymidine

(a

procedure

that

completely

inhibits cellular DNA

synthesis

without

affecting

viral DNA

synthesis [18])

and were

exposed

to

32p

to prelabel the nucleotide

pools.

The cellsweretheninfected with

PrV(1)33

and incubated in

BUdR medium

containing

32P.

(BUdR,

a

density label,

was addedtotheculturetoallow thedifferentiationbetween viral DNA that had

replicated

and viral DNA that had become labeled

by

repair synthesis only.)

At various times after

infection,

someof the cultureswere

harvested,

and the DNA was extracted and banded in CsCl

gradients along

with

[3H]thymidine-containing

and

[3H]BUdR-substituted

viral DNA as markers. DNA fractions were

collected,

and the

position

in the

gradient

of

32P-labeled

viral DNAwas deter-mined. The DNA

banding

with the

[3H]thymidine-labeled

viral DNA and the DNA that had shifted to a

position

of

higher density,

i.e., the DNA that had

replicated,

were collected

separately.

The DNA

preparations

were

purified,

digested with BamHI restriction enzyme, and

electropho-resed in agarose

gels.

Figure

2 showsthatBamHI

fragments

10aand 12b

[which

arethe

fragments

characteristic of

PrV(1)33-defective

DNA]

had

replicated

preferentially (lane 2) by

3 h 45 min

postin-fection. Even

though

these cells had been coinfected with

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Pr(1)24

A B

Pr(1)33

A B

1

_u

1 _

2 _ 2 _f _

3 - _ 3 _ _0

4 =.-.;.

6 I4 mm 4 _

7 - -l ,o5 4tU

5 ,

tu:r

6~~._

8 ~ ~ ~ ~ m 6 Mo

9~~~~~~~~~~~~~~~~~~~~~~

10 ~ ~ ~

1~~~~~~~ ~0

~~~~~~~~

.d

loa

a

1

t1 vY .). 1~~10 ; ]t 1 * 101

12

I

11I

.-:...;:.l* 12b

12b

FIG. 1. Comparison of the BamHl restrictionpatternsofvirion DNA with the total intracellular viral DNA. 32P-labeled virion DNA (A) andintracellular viral DNA (B)werepurified fromcells infected either with PrV(1)atpassagelevels24, 33, 43, and 64orwith standard PrV

[Pr(s)] asdescribed in Materials and Methods. An equal amountof32P-labeled DNA obtained from virions and from infected cellswas

digested with BamHI and electrophoresed inagarosegels, and autoradiogramswereprepared. The numbers onthe left side of each lane

designate the standard PrV BamHI fragments; the numbersontheright side designate the defective DNAfragments, i.e., the fragments that

arenotgeneratedby digestion of standardPrV DNA. Hindicatesaregion ofheterogeneity.

helper virus, fragments representative of the helper PrV

genome could barely be detected in the DNA that had

replicated (lane 2), i.e., had shifted to a position ofhigher

buoyant density as a result ofincorporation ofBUdR

(nor-mal thymidine-containing DIP DNA has a lower buoyant

density than doesstandard PrVDNA [4]). Inthis particular experimentasignificant amountof repair synthesishadalso occurred (lane 1);however, inotherexperiments much less repairsynthesiswas observed (data not shown). We donot know the reasonforthis variation.

Figure 3 shows the microdensitometer tracings of autora-diograms obtained from a similar experiment in which the

32P-labeled viral DNA that had accumulated in the infected cells at different times after infection was analyzed. The

results show that the defective DNA, exemplified by frag-ment 12b, replicated first and, in fact, was the prominent

species synthesized up to approximately 6 h postinfection. By 24 h postinfection, however, this band was much less

prominent,andtherestrictionpatternof the32P-labeled viral DNA that had accumulated in the cells resembled the restrictionpatternof the population of virionscontainingthe DIPs that had been usedtoinfectthe cells. We concludethat PrV(1)-defective DNA contains origins of replication that

are recognized at early times after infection by the DNA-synthesizing machinery of the infectedcells. Thereplicative advantage of the DIP DNA is,however, only transient.

The 32P-labeled DNAaccumulatingin cells infected with DIPs at various times after infection was also analyzed by

theSoutherntechnique(25).Themicrodensitometertracings of the hybridization pattern of this DNAtoKpnI fragments of standard PrV DNA fixed to nitrocellulose filters are

shown in Fig. 4.(See Fig. 6 formappositions ofrestriction

fragments.) Viral DNAsynthesized during the firstround(s) of replication hybridized exclusively to KpnI fragments B

and D (middle panel). (Further exposure did not reveal hybridization to any of the other KpnI bands.) By 24 h postinfection, the DNA synthesized by thecells hybridized toall KpnI fragments (toppanel), and the patternof hybrid-ization mimicked that of the DNA in the population of DIPs used for infection (see also reference 22). These results corroborate the conclusion that the DIP DNA replicates preferentially at early stages of infection in cells infected with a virion population which contains both DIPs and

helper virus [PrV(1)33].

Structural modification ofgenomesoftheinfectiousvirions

present in the populations of DIPs. The results described above indicate that although defective DNApresent in the population of DIPs has a replicative advantage (at early

stages ofinfection), itdoes not appearto posses an

advan-tagein encapsidation overthegenomesof the nondefective

virionspresentinthevirionpopulation (Fig. 1). The lack of preferential encapsidation of the defective DNA could be due to a lack of enrichment ofthis DNA in cis functions

required for encapsidation. On the other hand, it could be

dueto thepresence in thepopulation of DIPs of infectious

virus that has been modified and has acquired the abilityto compete effectively with DIPDNA, eventhough the latter has been enriched for cleavage-encapsidation sites. The

emergenceinapopulationofDIPs ofinfectious virionsthat,

as aresult ofsomemodification, haveacquiredtheabilityto

overcomethe selective advantages of the genomesofDIPs has been documented in other systems(10, 14-16).

To determine whether the genomes of the infectious

virions presentin thepopulation of DIPs ofPrVhave been modified, we analyzed (as a first step) the structure ofthe

genomes of individual plaque-purified infectious virions

ob-tained from the two independently derived populations of DIPs that we have been studying. Figure 5 illustrates the

Pr(1)43

A B Pr(1)64

A B

5.5

6 7

Pr(s)

A B

_UU

2 _&

-3_ _

4 -l _

56s l 7 _tWk

.8 _ _

98 _"f

9 _SO. _

10 w.

11 .,}- ---12

2. _

3 s m

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w ..

-.

-U.~~.

7 . ,

8 0o

8

or

9

10 -.

IOIF M._

1 1

', 10a

12

*

~~~~~~I

1

FIG. 2. Defective DNA replicates first in cells infected with a population ofPrV(1) DIPs.Cells wereincubatedin EDS-PO4+FU (toinhibit the synthesis of cellular DNA) and further incubated in EDS-PO4+FU containing32ptoequilibrate the nucleotide pools, as describedin Materials and Methods. The cells were then infected withstandard PrV or PrV(1)33 and were incubated in BUdR medium containing32pUp to3.75hpostinfection.The cells wereharvested, and the DNA waspurified by centrifugation inCsCI gradients, as described in Materials and Methods. The purified viral DNA was digested withBamHI and electrophoresed in agarose gels, and an autoradiogram wasprepared. Lanes: 1, viral DNAobtained from cells infected with DIPs that had banded in the characteristic position ofPrV DNA(6 days ofexposure); 2,viralDNAobtained from cells infected with DIPs that banded in a position of higher density (DIP DNA normally has a lower buoyant density than standard PrV DNA), i.e., that hadincorporated BUdR (6days of exposure); Pr, viralDNAobtainedfromcellsinfected with standard PrV that banded in a position of higher density, i.e., that had incorporated BUdR(2days ofexposure). M indicates the position of mitochondrial DNA, the synthesis of which is not completely inhibited by preincubation ofthe cells with FU.

Kpn digest of the DNA in the virions of10 representative

plaqueisolates obtained from PrV(1)42. Of interest was the

finding that in many cases a ladderlike series of submolar bands was observed; Kpnl end fragment D(Fig. 6), which normally ispresentinmolar amounts in standard DNA, was

in certain populations of virions either nondetectable or

considerably

reduced in

intensity.

Severalrestrictiondigests of the DNA of individual plaque isolates were analyzed by the Southern technique (25) with

individual cloned nick-translated restriction fragments

en-compassing the entire PrV genome as probes, and the genomes of these plaque isolates were mapped. The results showed that although only occasional modifications were observed along various parts of the genome of theseplaque isolates, more than 75% had genomes in which sequences

fromthe left end of the genome had become reiterated. The

ladderlike arrays of bands derived from the left end of the genome that were observed in the restriction digests were

probably the result ofunequal (out-of-register)

recombina-tion, a process that occurs when sequences are tandemly

reiterated.

Examples of the hybridization pattern of the BamHI

digestsof the DNA of someplaque isolatewith someprobes

of interest areillustrated in Fig. 7. Digestion of the DNAof

plaque isolate B7 generated a series of bands which hybrid-izedtocloned BamHIfragment 14' only (i.e., the left end of the genome; Fig. 6), indicating that sequences from the left

end of the genome, which did notincludethe BamHIcutting

site, hadbecome reiterated. On the other hand, digestion of the DNAof plaque isolate All generated a multimolarband

at approximately the same molecular weight as that of

BamHI fragment 14' that was generated from the standard virus DNA. This fragment hybridized to sequences present in BamHI fragment 14' only, indicating that the segment of DNA thathad become reiterated consistedofthe sequences ofBamHI fragment 14' as wellas some adjacentsequences

DIP

12 b I 3.5h

Z3

4 5.5:7 8,8'

9,10

11 12 13 14'

6

FlG. 3. Preferential replication of defective DNA up to 6 h postinfection. Cells were incubated in EDS-PO4+FU and further incubated inEDS-PO4+ FU,asdescribedinthelegendtoFig.2.The cells were then infected with PrV(1)33 and further incubated in EDS-PO4+FU containing 32p. The cells were harvested at the indicatedtimes,and the DNAwaspurifiedanddigestedwith BamHI andelectrophoresedin agarosegels. Autoradiogramswereprepared after an appropriate exposure time and scanned to illustrate the relative abundance of the various restrictionfragments.

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[image:4.612.326.515.293.645.2]

including theBamHI cleavage site. In other plaque isolates, as, for example, isolate A9, the reiteration not only com-prised the extreme left of the genome(i.e.,BamHIfragment 14'), but also included significant stretches of sequences derived from the adjacent BamHI 5' fragment. For isolate A9, the BamHI cleavage sites between the sequences in BamHI fragments 14' and 5' were lost. A multimolar

frag-menthybridizing to both BamHI fragment 14' and BamHI

fragment 5' was consequently formed. (Isolate A9 also contains inverted repeats of unequal sizes, as indicated by

thehybridizationpatternof itsDNAtoBamHIfragment 8'.)

Thus, the size of thefragment of DNA originating from the left endofthe genome that isreiterateddiffers in thedifferent

virus isolates. This may also be deduced from the increments in the number of nucleotides between the rungs of the ladder observed in theKpnI digest (Fig. 5). Indeed, in some cases,

KpnI band D appeared only as adiffuse smear. Analysis of the genomes ofmorethan 100 individual plaque isolates of

PrV(1)42 showed that the reiterations at theleft end ofthe genome varied in size from less than 300 to at least 1,500 base pairs (data not shown). The reiterations always in-cluded sequences derived from the extreme left end of the

B

D

B

DIP 24h

DIP

A B C D E FGCA- I J K

FIG. 4. Microdensitometer tracings ofthehybridization pattern

of viralDNAsynthesized by cells infected with PrV(1)33. Cellswere

infected with PrV(1)33, as described in the legend to Fig. 3, and harvested at the indicated times. The DNA was purified and

hybridizedtoKpnl restriction fragmentsofstandardPrVDNA that

had been fixed to nitrocellulose filter strips. The autoradiograms

were scannedafter appropriate exposure times. The hybridization

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patternof32P-labeled PrV DNAtotheKpnIfragmentsisalsoshown

[Pr(s)].

A21 A22 A23 A24 A25 A26 A27 A28 A29 A30

FIG. 5. KpnI restriction patterns of the genomes of different plaque isolates obtained from a population of virions containing DIPs. Individual plaques (A21 through A30) formed by infectious virions present in a population containing DIPs [PrV(1)33] were picked, and the virionswereamplified. ViralDNA waspurified and digested withKpnI. KpnIfragmentDisthefragment generatedfrom the left endofthegenome (see map in Fig. 6).

genome (BamHI fragment 14'). Similar results were also

obtainedwhenplaque isolates obtained fromPrV(2)42were analyzed (8).

The proportion of infectious virus genomes that had

reiterated ends in populations of DIPs was determined at several passage levels. Infectious virionspresentinPrV(1)at passages33, 43, and 53wereplaque purified,and 20plaques wereanalyzedin each case. No significant differences in the

relative numberof plaque isolates containinggenomeswith

reiterated ends were found; in all three cases, between 70 and85% ofthe plaques tested had genomes with reiterated

ends. Thus, the reiterations ofthe left end ofthe genome wereretained upon passageof the virions in the presenceof

DIPs. On the other hand, when individual plaque isolates

with reiterated end sequences were passaged in cell cultures in the absenceof DIPs, the genomes of some ofthe virions in the progenyacquired nonreiterated ends(Table 1). Thus,

it appears that the reiterated sequences atthe endoftheUL

donotconferonthe virionsanyreplicativeadvantage in the absence ofDIPs.

Underproduction of virions with nonreiterated end se-quencesby cellscoinfectedwith standardvirus and a popula-tion containing DIPs. Because

approximately

75% of the infectious virions present in the population of DIPs have

repetitive sequences at the left end of theirgenomes, these infectious virionscan be distinguished from standard infec-tious virions. It was possible, therefore, to determine whether standard virus would be amplified in unmodified

form as efficiently as was the endogenous infectious virus that was present in a population of DIPs when cells were coinfected with both.

Table2summarizes the results of this experiment. In the

original population of DIPs, PrV(1)42, 25 of 30 plaque

isolates (83%) had genomeswith reiterated ends. After the

population of DIPs was mixed with standard virions, the relative number of plaque isolates with genomes that had reiterated ends decreased, as expected, and only 6 of 30

plaque isolates (20%) were found to have reiterated ends.

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15 14 12

* 5- * 2 *9,11* 4 * 3 * 6 #6*'S6 5 *10 77 * 5 IS 13

L m N M

D * C *A JIGG * f Er K # J KiIt1 H

UL IRs U3 TR5

I s s|I s l k s s s & | t | ffi | s s IAA A aIIIaa IIa I I I I III a I A I iIII.1II

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

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MAP UNITS

FIG. 6. Restrictionmapsof the PrVgenome. Arrows indicate cleavage sites. Rectanglesrepresent theinvertedrepeats.

However,after thispopulation of virionswas passaged five

times at high multiplicity, most (21 of 29, or 72%) of the

plaque isolates hadreiterated ends, indicating that the stan-dard virus with thenonreiterated ends that had been added to the population of DIPs tended to be lost upon passage.

The disappearance of virus with genomes with standard nonreiterated ends observed in this experiment could be the result ofareplicative advantage of the virions with reiterated

ends thatareendogenouslypresentinthepopulationof DIPs

over standardvirions. On the otherhand, thestandard viral

genomes mayhaveacquired the reiterationsattheend ofthe

unique long(UL) segment as aresult ofrecombinationwith

thevirions presentin the population of DIPs.

To distinguish between these two possibilities, i.e., to ascertain whether in cellscoinfectedwithDIPs theDNA of standard viruscancompeteeffectively foramplification, we

compared (i) the relativeamounts of viral DNAsynthesized by cells either infected with standard virus alone or

coinfected with standard virus and DIPs that became

asso-ciated with mature virions (Table 3), and (ii) the relative

All

probes 5' 8 14' Pr

^.2

3

. 5'i

'I,j. 11 5

12

13

14'

87

5 8' 14 Pr

123

40

Ho

S& 0 Si

som

j 88

.* .11

12

amountsofparental standard PrV DNA that, when addedto cells infected with these virion populations, replicated and becameassociated with mature virions (Table 4).

The amount of viral DNA synthesized by cells infected with standard PrV alone and by cells coinfected with stan-dard and defective virions was approximately the same

(Table 3). However, the number of virionsproducedby the cells coinfected with the DIPs(as determined bytheamount of labeled DNA that became associated withpreparationsof purified virions) was reduced by almost 90%. Thus, in this experiment, 22.6% of the total viral DNA synthesized by cells infected with standard virus alone butonly 3.1% of the viral DNA synthesized by cells coinfected with DIPs and standard viruswasfoundinthepurified virion preparations.

This finding confirms previously published reports (2, 4) which showed that the number of virions produced by the cells coinfected withDIPs ofPrV is reducedconsiderably.

To determine whether standard viral genomes can

com-pete at the level ofreplication and encapsidation with the viral genomes in the population of DIPs, the fate of

B5C 5' 8 8' 14 Pr

1

3

mm 4

*_{_} .6a₇

0. 8

10

A9

5 8 8 2 14

a

4.

0

11

12

to 4

06

A* 7

-wa

8

9 10

12

13

A* 14

.<+d 1 3

13

4

FIG. 7. Hybridizationofsomenick-translatedBamHI restrictionfragmentsof the PrV standardgenometotheBamHI restrictiondigests

of theDNA ofdifferentplaqueisolates. Thegenomesoftheindicatedplaqueisolates(All, B7, B5C,andA9)weredigestedwithBamHIand

electrophoresed,andtheDNAfragmentsweretransferredtonitrocellulosepaper.Stripswereprobedwithcloned,nick-translated restriction

fragments originating fromthefragmentencompassing theentirePrV genome;however,the results obtained withsome of the probes of

interestonly(BamHI fragments 5', 8',and 14') arepresented. Because of theheterogeneityofsomerestrictionfragments, allofthe viral

isolates inthisexperimentwereplaque purified again;the results obtained wereidentical.

14

Bus HI

Kpa

0 A& 14'+5'

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TABLE 1. Reversion ofvirionscontaininggenomeswith

reiterated endsto virions withnonreiterated end fragmentsa No. of virions

withgenomes

Plaqueisolate containing

standard nonreiterated

ends

B1... 0/8"

B5... 6/9

B7... 2/9

B12... 0/3

All ... 9/12

aTheprogenyofsomeindividual plaque isolates obtained from populations

ofDIPs which containedgenomeswithreiterated ends (Fig. 7)waspassaged

fivetimes in RKcellsatlowmultiplicity andthenplaqueassayed.Individual

plaques were picked, the virions were amplified, and theirgenomes were

analyzed with restriction enzymes. The number ofplaque isolates which

contained DNAwith standard nonreiterated endswasdetermined.

bNumerator, number of plaques yielding virions with genomes with

standardends;denominator, number of plaque isolates analyzed.

[3H]thymidine-labeled parental standard viral DNA in cells coinfected witheither unlabeled standard virus orunlabeled

DIPs was determined (Table 4). This experiment was

per-formed underexperimental conditionsthatwereidentical to those used in the experiments summarized in Table 3. The results of a representative experiment are summarized in

Table 4. This experiment was repeated three times with

essentially similar results.

Both in cellsinfected with standard virus alone and in cells coinfected with DIPs, most of the parental standard viral DNA had replicated by 8 h postinfection. The amount of 3H-labeled parental standard virus DNA that became

asso-ciated with progeny virions produced by cells coinfected with DIPs was reduced approximately 10-fold compared

with that produced by cells infected with standard virus alone (Table 4, column 4). Sinc,e the total number of virions produced by these infected cells also differedapproximately

10-fold (Table3), onemight conclude that the standard and

defective genomes compete equally well forencapsidation.

However, a large proportion of the [3H]thymidine-labeled

parental virus DNA that was present in the population of

progeny virions obtained from cells coinfected with DIPs

was DNA that had notreplicated in the infected cells (Fig. 8). This DNA was probably derived from input parental virions that hadnotbeen uncoated and thatcopurified with

theprogeny virionsproduced by the cells. Ifonecompares

TABLE 2. Loss ofvirions withgenomeswithstandard ends from

apopulationcontaining DIPs'

Beforepassage Afterpassage

No. of No. of

Virionpopulation genomes genomes

PFU/ml with PFU/ml with reiterated reiterated

ends ends

PrV(1)41 1.3 x 107 25/30 1.3 x 107 24/28

PrV(1)41 + 4.1 x 107 6/30 1.7 x 107 21/29

standard PrV

ToapopulationofDIPs[PrV(1)411,sufficientstandard PrV wasaddedto

increase the titerofinfectious virusapproximatelythreefold.Part of thevirion

populationwasplaqueassayed,andpartwaspassagedfive times inundiluted

form andthenplaque assayed. Thirtyindividual plaquesfrom eachvirion

population were picked; the virions were amplified, and the DNA was extractedandanalyzedwithrestrictionenzymesto determine thetypeof end

[image:7.612.328.569.106.159.2]

fragments (standardorreiterated)thatwerepresent.

TABLE 3. Synthesisandencapsidationintovirionsofviral DNA in cells infected with standard PrV aloneorin cells coinfected

withstandard PrV andDIPsa

3H-CdRin 3H-CdRin ViaDN

Cellsinfected with: viral DNA purifiedvirions al DNA

(103cpm) (103 cpm) invirion (%)

PrV 97.5 21.9 22.6

PrV + PrV(1)42 73.2 2.8 3.1

a RK cells (4 x 106persample) were incubated in EDS-FU for 16 h (to

inhibit cellular DNA synthesis) and were then infected with either PrV alone

(5PFU percell)or amixture of DIPs(approximately300particles;2PFU per

cell)and standardPrV(3 PFU percell).Unadsorbed virus was removed after

1h, and the cellswereincubated in BUdR mediumcontaining 3H-CdR(50

RCi/ml)for 48 h.The totalamountof3H-CdRincorporatedinto viralDNA,as

wellasthe amountof3H-CdR-labeledDNA that hadbecomeencapsidated

intomaturevirions, was determined as described in Materials and Methods.

the amount of3H-labeled parental standard DNA that had

replicated and become part of virions produced by cells coinfected with DIPs with the amount produced by cells infected with standard virus alone, one is led to conclude that thestandard DNAwasdiscriminatedagainstatthe level ofencapsidationin cellscoinfected with DIPs.Thus, incells infected with standard virus alone, the same proportion of the total viral DNA (22.6%) and of the 3H-labeled parental

viral DNA that hadreplicated (24.0%) becameencapsidated

into virions (see Tables 3 and 4). In cells coinfected with

DIPs, 3.1% of the total viral DNA but only 0.9% of the 3H-labeled parental viral DNA that had replicated became encapsidated. These results show that standard viral DNA is discriminated against at the level ofencapsidation in cells coinfected with DIPs.

Figure 1 shows that the defective DNA does not appearto be preferentially encapsidated overthe nondefective DNA endogenously present in the population of DIPs. On the otherhand,Tables 3 and 4 indicate thatexogenously added standard PrV DNA is discriminated against at the level of encapsidation in cells coinfected withamixture of DIPs and thestandard virus.Together, these findings indicate that the infectious virions present in the population of DIPs are modified forms of the standard PrV genome in thatthey have acquired characteristics which allow themtocompete

effec-tively with DIPs for encapsidation. Furthermore, these results indicate that the defective DNA isprobably enriched forcleavage-encapsidationsignals, despite the apparent lack ofpreferential encapsidation of the defective DNAoverthe nondefective DNA present in the virion population.

DISCUSSION

The salient features of the results presented in this paper can be summarized as follows. In a population containing DIPs ofPrV, the defective DNA has a replicativeadvantage atearly stages of DNAreplication. The defective DNA does not, however, appear to have an advantage at the level of

cleavage-encapsidation and is not preferentially encapsid-ated over nondefective viral DNA present in the virion population.Despite the lack ofpreferentialencapsidation of the DIP DNA, it appears that this DNA is nevertheless enriched forcleavage-encapsidation sites. This conclusion is based on thefollowing observations. (i) The genomes of the infectious virions present in the population of DIPs have beenaltered;notably reiterations of sequences derived from the left end of the genomearepresentinapproximately75% of the genomes of these virions. (ii) While standard viral genomes arediscriminated againstat the levelof

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TABLE 4. Replication and encapsidation of13H]thymidine-labeledstandardparental viral DNA in cellscoinfected withDIPsa 3H-Pr(s)b 3H-Pr(s)DNAthat [3H]Pr(s)_{in progeny}DNA [3H]PrV DNA in_{progenyvirions} [3H]PrVDNA in

Cellscoinfected with: DNA in cells had replicated by 8 h irons tahdepicated progenyvinons that

(103cpm) (103

cpm)

(13o

cpm)

(103cpm)

hadreplicated (%)

(3H)PrV(S) + PrV(S) 27.6 19.55 5.49 4.70 24.0

(3H)PrV(S)+ PrV(1)42 22.2 14.16 0.59 0.13 0.9

aCells were coinfected with purified[3H]thymidine-labeled virions(3 PFU per cell)and either standard or defective unlabeled virions (2 PFU per cell). After 1

h, unadsorbed virus was removed and the cells were further incubated in BUdR medium. At 8 h postinfection, a portion of the cultures was harvested and the

DNAwasextracted and centrifuged in aCsCI gradient with 32P-labeled thymidine-containing or BUdR-substituted DNA as markers to determine the amount of

parental virus DNAthathadreplicated (column 3). The remainder of the cultures was incubated up to 48 h postinfection, when the virions produced by the cells

were purified. The total amount of parental3H-labeledPrV DNA(column 4) as well as the amount of DNA that had replicated in the infected cells (column5; see

also Fig. 8) that were transferred to the progeny virions was deterined.

bPr(s), StandardPrV.

c P.i.,Postinfection.

tion in cells coinfected with DIPs, the nondefective and

defective genomes present in these populations of virions

compete equally well for encapsidation. It appears,

there-fore, that both the nondefective and defective genomes in these viral populations have an advantage over standard virus at the level of encapsidation and probably are enriched

for cleavage-encapsidation sites.

Our results showthat thedefective DNAreplicates firstin

the infected cells when the synthesis of the nondefective virus DNA present in the population of DIPs is barely detectable. Therefore, the defective DNA in thepopulation of virions studied is

probably

enriched for sequences that can serve as

origins

of

replication,

sequences that can

function at early stages of infection and compete

success-fully with the origins present in the nondefective DNA. Vlazny and Frenkel (29) showed that the defective DNA present in a population of DIPs of HSV is synthesized mainly at late times after infection, i.e., is enriched for origins of

replication

thatdonotfunction wellatearlystages

12.8 Pr

I+H L-L

9.6-

6.4-CY 3.2

0

a.i 3

Pr(1)42

3-

2-20 30 40 50

[image:8.612.52.551.93.153.2]

FRACTION NUMBER

FIG. 8. Analysis ofthe3Hparentalviral DNA strands thatwere transferredtothe virus progenyproduced by cells coinfected with DIPs. RK cells were coinfected with either standard PrV or PrV(1)42 and [3H]thymidine-labeled standard PrV, asdescribed in thefootnotetoTable 4. The DNAoftheprogeny virionsproduced bythe cells (Table4, column 4) was centrifugedtoequilibrium in CsCl, together with 32P-labeled thymidine-containing DNA and 32P-labeled BUdR-substituted viral DNA as markers. Fractions were collected, and the distribution of label in the gradient was determined.The bottomofthegradientis at the left.

of infection. The differences between our results with PrV DIPsandthose of Vlazny and Frenkel (29) with HSV DIPs are probably due to an enrichment of different types of

sequences that can serve as origins of replication in the genomes ofthe two populations ofDIPs; it is unlikely that they reflect intrinsic differences between these two herpesviruses.

Normally, during the first roundof replication, PrV DNA

synthesis is initiated mainly at a site near or within the

inverted repeats(7). Southern hybridization analysis ofthe DNAsynthesized duringthefirstround ofreplication in cells infected withDIPsindicates that thesequences thatreplicate firstdo notincludesequenceslocated intheinvertedrepeats

(Fig. 4). These results might indicate that an origin other

than that intheinverted repeatsispresent in the DIP DNA

and is recognized at early stages of infection. However, because recombinational events occurredduring the

gener-ation of theDIPDNA,wecannotexcludethepossibility that the

origin

normallypresentinthe invertedrepeatshas been

juxtaposed (by a recombinational event) next to the se-quencesin theDIP DNA thatreplicate

preferentially

atearly

stages of infection. The presence of a small stretch of

sequencesderivedfrom the invertedrepeatswouldnothave been detected inourexperiment. Thus, althoughoutresults show that defective DNA is enriched for an origin of replication that canbe usedat early stagesofinfection, this origincannotbeconclusively identified inthe absenceof the appropriate sequencing data. The reason for the transient preferential synthesis only ofthe DIP DNAis alsonotclear.

As mentioned above,although ourresults show that DIP DNA replicatespreferentially under certain conditions,it is not, as far as we can tell, preferentially encapsidated. However, the lack ofpreferential encapsidation of the DIP DNA does not necessarily reflect a lack ofenrichment for

thecleavage-encapsidation site. Ithas beenestablishedthat the dynamics of the interactions between the DIPs and

infectious virions present in a population containing DIPs areaffectednotonlybythesequencecomposition ofthe DIP DNA but alsoby mutational changes in the genomes ofthe

endogenous infectious virions (10,

14-16).

Indeed, our re-sults show that a selection for infectious virions that have

acquired

characteristics

that promote their ability to com-pete with the DIP DNA has occurredduringtheevolution of thepopulation ofDIPs. Mostoftheseinfectiousvirions have

acquiredreiterations of theleftendofthegenome

(Fig.

5and 7); these reiterations tend to be lost upon passage of the virions in the absence of DIPs (Table 1). Furthermore, infectious virions containing standard PrV DNA with nonreiterated ends are discriminated against in cells coinfected with DIPs(Table 2), andalthough the DIP DNA and the endogenousnondefective DNA present in the DIP

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population compete equally well (Fig. 1), standard PrV

genomes compete poorly at the level of encapsidation in cells coinfected with apopulationofDIPs (Tables 3 and 4). Taken together, these dataindicate that both the defective DNA and thegenomesof the infectious virionspresentin the population of DIPshaveanadvantage in encapsidationover

the standard viral genome.

The segments of DNA derived from the left end ofthe

standard viral genomethatare reiterated in thegenomes of infectious virionspresentin thepopulation ofDIPs contain someofthe cisfunctions thatare necessary forthe efficient

cleavage-encapsidation of PrV DNA (30; C. Wu, Ph.D thesis, Vanderbilt-University, Nashville, Tenn., 1986). The possibility that the reiteration of these sequencesis linkedto the advantage of these genomes in encapsidation is attrac-tive. However, conclusive evidence that these reiterations

are the basis for the selective advantage of the modified

virions is not available; other, undetected, modifications in thegenomes may play a role.

The cyclical changes in the level of infectious virus obtained upon passage of populations ofDIPs at high mul-tiplicity that are observed in many other viral systems are

not observed upon similar passage of PrV DIP. Further-more, the accumulation of the DIP DNA in this system is slow (4). The reason for these characteristics is probably

related to the fact that the DIPs only have a slight growth

advantageoverthe infectious viruspresentin thepopulation of DIPs that we have studied. Their genomes have only a

transient replicative advantage at early stages of infection (Fig. 2 and 3), and a significant advantage at the level of encapsidation of DIP genomes over the genomes of the

infectious virions present in the population of DIPs is also notobserved (Fig. 1). Asalready mentioned,this is probably because modifications of the genomes of the endogenous

infectious virions have occurred that allow them tocompete

forencapsidation.

Despite the fact that the DIP DNA has only a slight

replicative advantage, the populations ofDIPs do interfere significantly with the growth of standard virus (2). This

interference can be ascribed to apreferential encapsidation

of the genomes present in the DIP population over that of

standard virus. It is also due to an interference with the

process of cleavage and encapsidation of viral DNA

(stan-dardas wellasdefective) in DIP-infected cells (Tables 3 and

4). Thus, even though viral protein synthesis (22) and the

production of capsids(unpublishedresults)arenotmarkedly affected,thetotalnumberof viral particlesproducedand the cleavage of concatemericDNAis reduced byapproximately 90% in cells coinfected with DIPs (4). The reduction in cleavage and encapsidationof the virusmaybethe result of

an interference by the DIPs with the orderly expression of

the virus genome. On the other hand, it may be due to the direct inhibitory effect ofthe DIPgenomes on the

cleavage-encapsidation process as a consequence of the sequence

organization of the DIP DNA. This question remains to be clarified.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Servicegrant

AI-10947from theNational InstitutesofHealth.

Weappreciate theexcellent technical assistance of PatComers.

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